Urotensin II Enhances Advanced Aortic Atherosclerosis Formation and Delays Plaque Regression in Hyperlipidemic Rabbits

Figures

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  Figure 1. UII infusion aggravates atherosclerosis in ovariectomized female rabbits. (A) Study design: UII (5.4 μg/kg/h) or saline was infused via osmotic pumps into ovariectomized rabbits with a 0.3% cholesterol diet for 12 weeks. The en face lesions were visible after Sudan IV staining. (B) Quantifications of the lesional area of the whole aortic tree. (C) Representative images of immunohistochemical stainings. (D) Estrogen levels of sham and ovariectomized female rabbits. (E–H) Quantifications of immunohistochemical staining. All data are expressed as mean ± SEM. All data were tested for normality and equal variance. If passed, one-way analysis of variance (ANOVA) followed by Tukey post hoc test for comparisons among >2 groups. Otherwise, nonparametric tests (Kruskal–Wallis test followed by Dunn’s post hoc test) were used. n = 6–10 per group. * p < 0.05 and ** p < 0.05 vs. vehicle group.

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  Figure 2. Schematic illustration of the advanced plaque experiment design and gross lesion analysis. (A) Rabbits were fed an HCD and were infused with either UII (5.4 μg/kg/h) or saline via osmotic mini-pumps for 24 weeks (left panel). Aortic trees along with the heart were collected and divided into 4 parts for the following analysis: coronary plaque analysis (1); an aortic ring for calculating plaque vulnerability index (2); remaining aorta tree for analyzing aortic lesions (3), and carotid and subclavian arteries of the aortic branches (4) (right). (B) The representative images of Sudan IV-stained aortas from each group (left panel). Quantification of the sudanophilic area of the whole aortas was shown (right panel). (C) Microscopic analysis of the aortic lesions. Representative micrographs stained by H&E, EVG, collagen, macrophages (Mφ), SMCs, and Oil red O from each group (upper panel). Quantifications of microscopic atherosclerotic area, collagens, Mφ, and SMCs and vulnerability index of aortic arch plaques (lower panel). * p < 0.05, ** p < 0.01 vs. vehicle group. (D) Analysis of intra-plaque angiogenesis. Neovessels in the aortic plaques were detected by CD31 staining (arrows). The…

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  Figure 3. Analysis of coronary atherosclerosis. (A) Schematic illustration of heart sectioning procedure. (B) Calculation method of coronary atherosclerotic lesions. (C) Representative figures of coronary atherosclerosis of seven blocks. The sections were stained with H&E stain of each group. Quantification of coronary arterial stenosis rate (%) (left panel). * p < 0.05, ** p < 0.01 vs. vehicle group. (D) The lesions (blocks III and VI) were either stained with Masson trichrome for collagen or immunohistochemically stained with anti-Mφ Ab (left panels). Positively stained areas were quantified (right panel). n = 3–10/group, all data are presented as mean ± SEM.

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  Figure 4. Analysis of the lesions of the common carotid and subclavian arteries. (A) Analysis of gross lesion areas. Representative images of four branches (containing right subclavian and common carotid arteries, left subclavian and common carotid arteries) stained with Sudan IV (left panel). The plaque area distributed within 1 cm extending upward from the root was measured and the total areas of four branches were shown (right panel). (B) Analysis of the microscopic lesions. These branches were stained with either H&E or EVG stain (left panel). The lesion size was measured using EVG-stained sections (right panel). n = 10/group, all data are presented as mean ± SEM. * p < 0.05.

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  Figure 5. UII delays the regression of atherosclerosis in rabbits. (A) Study design: advanced atherosclerosis was induced by HCD feeding for 24 weeks, then changed to chow diet for 8 weeks recovery for plasma cholesterol decreased to a normal level, and UII (5.4 μg/kg/h) or saline was infused for another 12 weeks. (B) Representative images of aortas after Sudan IV staining and their gross lesional quantification. (C) Representative microscopic images and quantifications of immunohistochemical staining. All data are expressed as mean ± SEM. n = 5–6 per group. ** p < 0.05 vs. vehicle group.

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  Figure 6. Determination of UII effects on the cultured cells. (A) NOX2 expression after being treated with 100 nM UII with or without urantide for 24 h in cultured macrophages. (B,C) ROS synthesis in macrophages was determined by the detection of the DCFH-DA fluorescent intensity. Representative images were shown in (B) and fluorescent intensity was quantified and shown in (C). Scale bar, 100 μm. (D) qRT-PCR (left) and WB (right) analyses of HIF-1α/VEGF-A expression in macrophages after UII with or without urantide treatment for 24 h. UII treatment significantly increased the expression of these two genes but was inhibited by the presence of urantide. Western blotting confirmed that UII upregulated HIF-1α/VEGF-A expression and this upregulation could be partly blocked by urantide. (E) and (F) HIF-1α expression in macrophages was detected by fluorescence microscopy and green fluorescence was used to label HIF-1α, scale bar, 50 μm (E). UII upregulated HIF-1α expression and could be partially blocked by urantide. Quantification of the relative intensity of HIF-1α fluorescence (F). (G) and (H) UII stimulated angiogenesis in Matrigel with seeded HUVECs after 4 h of culture. Quantification of…